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Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

Abstract

Background

Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.

Results

EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 102 to 1.3 × 108 copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 103 to 2.0 × 105 copies/ml in infectious mononucleosis (n = 7), 7.5 × 104 to 1.1 × 105 copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 102 to 5.6 × 103 copies/ml in HIV-infected patients (n = 12), and 2.0 × 102 to 9.1 × 104 copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).

Conclusion

Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.

Background

Epstein-Barr virus (EBV) causes infectious mononucleosis, an acute but self-limiting disease affecting children and young adults. After primary infection, the virus persists indefinitely in B-lymphocytes [1], only to reactivate when cellular immunity is impaired. In immunocompromised individuals, EBV-related disorders following virus reactivation are associated with significant morbidity and mortality [2]. Up to 15% of transplant recipients develop post-transplant lymphoproliferative disease (PTLD), a heterogeneous group of disorders characterised by EBV transformation of lymphocytes [3, 4]. Although uncommon, PTLD is aggressive and coupled with high mortality rates of 50-80% [4]. Also related to other diseases in immunosuppressed individuals, including chronic active EBV, fatal infectious mononucleosis (IM) and EBV-associated haemophagocytic syndrome (EBVAHS) [5–7], EBV is linked to several malignancies such as nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma (BL) [5]. In HIV-infected individuals, EBV is associated with diseases such as oral hairy leukoplakia and AIDS-related non-Hodgkin's lymphoma [5, 8].

Though sometimes detectable in the immunocompetent [9], EBV DNA is found in greater concentrations in immunosuppressed populations [10–13]. The presence of circulating EBV DNA does not always correlate with symptomatic infection, nor does it predict clinical disease in immunocompetent or immunosuppressed individuals [2, 9]. Nevertheless, although the correlation between EBV burden and disease status is incompletely understood, several studies have shown an association between symptomatic infection and elevated DNA loads in clinical samples [14, 15]. Increasing virus burden is also believed to be a rapid indicator of immunopathological changes preceding and/or underlying the B-lymphocyte driven changes caused by EBV [16]. Therefore, determining EBV DNA loads in EBV-related disorders in immunocompromised populations is an important step towards disease diagnosis, management and treatment [17].

Several methods for quantifying absolute DNA load have been developed since its first application to EBV diagnostics in 1999 [18–20]. These include semi-quantitative, quantitative competitive and real-time PCR methods [21], with each using different means for amplicon detection; visualisation on agarose gel, Southern blot analysis and enzyme immunoassay [21]. Real-time PCR quantification is generally preferred for its wider dynamic range, speed, ease of handling, sensitivity and specificity [2, 22–25]. Although commercial assays incorporating probe-based chemistries are available [26, 27], in-house methods employing high saturating dyes such as SYBR Green I are more cost-effective and just as sensitive as the widely used TaqMan PCR [21, 28–30].

Here, in an effort to ascertain the relationship between EBV DNA load and disease, two real-time quantitative PCR (QPCR) assays using SYBR Green I dye and a single quantification standard incorporating two separate EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed. EBV DNA was measured in a range of clinical samples, including unfractionated whole blood, plasma, PBMC and CSF from patients with EBV-associated disorders or immune dysfunctions. EBV sero-status was also determined for individuals in a population sample to assess the correlation between DNA load and antibody titres.

Methods

Groups with EBV-associated diseases or immune disorders

A total of 60 clinical samples from 25 individuals with various EBV-associated diseases or immune disorders were collected between February 2007 and September 2008. Specimen types included EDTA whole blood, plasma, PBMC and CSF. Each patient was assigned a letter (A to Y) and classified into one of four groups. Group 1 consisted of five patients with EBV-related PTLD following matched-unrelated donor haematopoietic stem cell transplantation, generating 40 samples: whole blood (n = 20), plasma (n = 18) and CSF (n = 2). Group 2 consisted of seven patients with IM, with plasma (n = 4) or whole blood (n = 3) samples and Group 3 was based on a single patient with EBVAHS from whom a whole blood sample was available. Group 4 consisted of PBMC (n = 3) and plasma (n = 9) samples from 12 HIV-infected individuals with HIV RNA plasma loads greater than 10,000 copies/ml.

Population sample

A fifth group was comprised of 218 individuals from a population sample for whom whole blood and serum were collected between 2004 and 2007. This included 46 males and 172 females with a mean age of 39 (SD = 10) and 40 (SD = 9.5) years respectively. These individuals resided in one of four regions in eastern Australia including Brisbane (n = 78), Newcastle (n = 28), Geelong and the western districts of Victoria (n = 45) and Tasmania (n = 67) [31].

Serology testing

EBV-specific antibody detection in the population sample

Quantitative EBV-specific serology was performed on sera from individuals in Group 5 only. EBV VCA IgG antibodies titres were determined by an immunofluorescence assay (IFA) using FITC conjugated anti-human IgG prepared in goats (Sigma-Aldrich, Castle Hill, NSW, Australia). Cells from the B95-8 marmoset cell line productively infected with EBV were grown in 27 mls of RPMI 1640-modified (ThermoFisher Scientific, Scoresby, VIC, Australia) +10% foetal calf serum (FCS) (ThermoFisher Scientific, Scoresby, VIC, Australia) medium containing 3 mls of 0.4 mM phosphonoacetic acid (Sigma-Aldrich, Castle Hill, NSW, Australia). Cells were spotted on 10 well slides (Pathech, Preston, VIC, Australia) and used as the antigen. Four-fold dilutions of known EBV positive sera were used as controls. Samples were diluted using phosphate buffered saline containing 10% FCS four-fold from 1:10 to an endpoint; samples with a titre < 1:10 were reported as negative, whilst titres equal to or greater than 1:10 were defined as positive.

Molecular testing

EBV gene targets, beta-globin and PCR controls

To maximise detection rates and reduce false negative results, two primer sets targeting the highly conserved EBV regions, EBNA-1 and BHRF-1, were used for PCR amplification (Table 1). EBNA-1 is a latent protein required for replication and genome maintenance and is the only viral protein consistently expressed in EBV-infected cells [32, 33]. BHRF-1 is expressed in lytic infection and confers anti-apoptotic properties similar to Bcl-2 for enhancing cell survival [34]. Groups 2-5 were evaluated by both PCR targets, while inadequate sample volume limited testing to EBNA-1 in Group 1. The beta-globin gene targeting the TAL57 region was used as a 'house-keeping' gene to control for PCR inhibitors and check for DNA integrity [35]. All samples were subjected to beta-globin PCR prior to EBV QPCR. Contamination was monitored by the use of PCR-grade water and no template DNA controls.

Table 1 Oligonucleotides used for EBV QPCR, beta-globin detection, construction of plasmid and PCR thermal cycling conditions.

DNA extraction and molecular assay design

DNA was isolated from 200 μl of EDTA whole blood, plasma or CSF using the GenElute™ Mammalian Genomic DNA Miniprep Kit® (Sigma-Aldrich, Caste Hill, NSW Australia) according to the manufacturer's instructions, and eluted in 200 μl elution buffer. The QIAamp DNA mini kit (Qiagen, Doncaster, VIC, Australia) was used to extract DNA from PBMC in accordance with the manufacturer's instructions. Extracts were aliquoted in single use volumes to prevent freeze-thaw cycles and stored at -80°C prior to testing. Each reaction mixture was contained in a PCR-certified colourless 200 μl flat capped tube (Integrated Sciences, Willoughby, NSW, Australia) to a final 25 μl volume, comprising of 2.0 μl LightCycler® FastStart DNA Master SYBR Green 1 dye (Roche Diagnostics, Castle Hill, NSW, Australia) at 10× concentration pre-combined with the LightCycler® FastStart enzyme, 0.5 μl of 0.2 mM sense and antisense primers (Invitrogen, Mount Waverley, VIC, Australia), 0.8 μl of 25 mM MgCl2 and 5 μl of the DNA eluate. Samples were tested on the 36-well rotor on the Rotor-Gene 6000® analyser (Qiagen, Doncaster, VIC, Australia). PCR was divided into two cycles: a first cycle with three repeats at 40 seconds for each stage, and a second cycle with 40 repeats at 30 seconds per stage. Thermal cycling conditions included an optimised initial denaturation step followed by 95°C denaturation, optimised annealing temperatures and extension at 72°C (Table 1). To ensure complete product formation, a final extension step at 72°C for 5 minutes concluded the PCR. A melt analysis immediately followed at between 60°C to 99°C as a check for amplicon purity. For confirmation, EBNA-1 and BHRF-1 products were electrophoresed in 2% agarose gel containing 1:20 dilution of SYBR® safe DNA gel stain in 0.5× TBE buffer (Invitrogen, Mount Waverley, VIC, Australia).

Cloning of EBNA-1 and BHRF-1 DNA targets into plasmid vector pGEM and standard curve construction

A novel feature of the assay was the design of a quantification standard incorporating both EBNA-1 and BHRF-1 DNA targets in a single plasmid (Figure 1). This was done to minimise the necessity for two separate EBV standards, thus reducing costs and labour. The EBNA-1 and BHRF-1 DNA targets were linked using randomised primers (Table 1) and inserted into the pGEM vector, using the pGEM®-T Easy Vector System II (Promega Corporation, Alexandria, NSW, Australia) according to the manufacturer's instructions. The cloned targets were then purified using the PureYield™ Plasmid MidiPrep System (Promega Corporation, Alexandria, NSW, Australia), and stored in single use aliquots. Target copy number was calculated following double stranded DNA approximation using the Beckman DU® 530 Life Science UV/Vis spectrophotometer (Beckman Coulter, Gladesville, NSW, Australia). A new plasmid aliquot was used for standard curve dilution for each PCR run consisting of three replicates starting at 101 to 106 copies/5 μl. PCR runs were accepted when the standard curve correlation co-efficient was ≥ 0.99.

Figure 1
figure 1

Plasmid vector pGEM showing location of cloned insert.

Product identification, reproducibility, sensitivity, limit of detection and specificity

PCR products were identified by an amplification curve, melt analyses and amplification efficiency generated by the Rotor-Gene™ 6000 Software 1.7 (Build 90). Positive EBV DNA samples had a cycle threshold (CT) less than 40, and melted between 86°C to 87°C with an average amplification efficiency of 1.74. PCR products for EBNA-1 DNA and BHRF-1 DNA were identified on agarose gel by 213 bp and 208 bp bands, respectively. Reproducibility studies consisting of triplicates of each standard curve dilution (101-105 copies/5 μl) were performed prior to testing. Intra-assay variation was determined in three repeat assays tested within 24 hours on three consecutive days. Inter-assay variation was assessed using three different batches of the same PCR master mix kit. Sensitivity was determined by end-point PCR using gel electrophoresis. To establish the minimum DNA copy number that could be reliably detected, ten plasmid replicates spanning 100 to 102 copies/5 μl were assayed in three separate runs. Primer specificity was verified on the Basic Local Alignment Search Tool on GenBank and by assaying known cytomegalovirus (CMV), human herpesvirus 6 (HHV6), HIV and varicella zoster (VZV) positive samples. The EBV QPCR was evaluated against an external quality assurance program (Quality Control for Medical Diagnostics (QCMD), Glasgow, Scotland, http://www.qcmd.org/ for EBV QPCR in 2008 and 2009.

Viral load calculation and result interpretation

Viral load calculations were based on DNA extraction volume and final elution volumes as well as the number of replicates tested. Samples were extracted and eluted in equal quantities, keeping ratios constant. Hence, the amount of sample used for PCR (5 μl) was multiplied by a factor of 200 (elution volume) and divided by the number of replicates to obtain a final measurement expressed as DNA copies per millilitre (copies/ml) of sample. This unit of measurement has close correlations with copies per microgram of DNA, therefore does not require normalisation to the amount of input DNA [36]. Furthermore, copies/ml removes unnecessary processing steps and reduces costs, as well as minimising sample volume for testing. EBV DNA was quantifiable in a dynamic range spanning six logarithms with the minimum reportable viral load at 2.0 × 102 copies/ml of sample. Samples with no detectable target DNA were assigned a load of zero and resulted as negative.

Statistical calculations

Data analysis was conducted with SPSS version 17. Spearman's (rho) correlation co-efficient was used to assess the correlations between EBNA-1 and BHRF-1 DNA loads and VCA IgG antibody titres.

Results

Performance of EBV QPCR assays: reproducibility, sensitivity, detection limit and specificity

The intra-assay and inter-assay co-efficient of variation for EBNA-1 and BHRF-1 QPCRs are shown in Table 2. Both EBV targets were detected at levels as low as 2.0 × 102 copies/ml of sample. However, the reliable limit of detection for both EBNA-1 and BHRF-1 DNA was 2.0 × 103 copies/ml, where the proportion that were detected (positivity ratio) were 97% and 93% respectively. Primers showed no cross reactivity to other herpesviruses (data not shown). All samples in both the 2008 and 2009 QCMD programs were correctly identified using the EBNA-1 primers.

Table 2 Intra- and inter-assay co-efficient of variation for EBNA-1 and BHRF-1 QPCRs.

EBV detection and load in EBV-associated disease states and immunocompromised individuals

Of the 60 samples from 25 immunocompromised patients, 30 (50%) samples from 16 (64%) patients had quantifiable viral load using one or other of the EBV DNA targets, EBNA-1 or BHRF-1 (Table 3). EBV DNA was detected in 100%, 85.7%, 100% and 33.3% of patients with PTLD, IM, EBVAHS and HIV-infected individuals (Groups 1 to 4), respectively. EBV DNA loads were detectable at ranges from 2.0 × 102 to 1.3 × 108 copies/ml in these clinical samples, with the highest EBV DNA load recorded in an individual with PTLD (1.3 × 108 copies/ml of sample). High levels were also seen in individuals with IM (2.0 × 105 copies/ml of sample), EBVAHS (1.1 × 105 copies/ml whole blood), and HIV infection (5.6 × 103 copies/ml of sample).

Table 3 EBV DNA loads in various EBV-associated disease states and immunocompromised individuals.

In Group 1 (PTLD), EBV DNA concentrations spanned six logarithms and were detected in multiple samples from early to end-stage disease. EBV DNA loads increased sequentially following transplantation, decreased after anti-viral therapy in Patients A and C and peaked ten days prior to death in Patients A to D. EBV DNA loads were detectable in some samples, but were absent in others. In Patient D, plasma EBV DNA was qualitative PCR negative on Day +62 whilst simultaneously QPCR positive in whole blood. EBV-specific serology results were available for four patients, and confirmed EBV infection prior to the transplant. Four patients died as a result of PTLD complications, on average +81.25 days post transplantation. In Group 2 (IM), EBV DNA was quantifiable from 1.5 × 103 to 2.0 × 105 copies/ml. One sample was negative for EBV DNA (Patient G), despite a positive EBV VCA IgM profile. Group 3 (EBVAHS) EBV DNA load results were similar to Group 2, however Patient M died as a consequence of the disease condition. In Group 4 (HIV), EBV DNA was detectable in both plasma and PBMC ranging from 2.0 × 102 to 5.6 × 103 copies/ml. However, 50% of these samples were below 2.0 × 103 copies/ml.

EBV detection and load in the population sample

EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of Group 5 (the population sample), respectively; 22.5% of samples were positive for at least one EBV DNA target (Table 4). Of the 24 EBNA-1 DNA positive samples, 91.7% were also BHRF-1 DNA positive, and of the 47 BHRF-1 DNA positive samples, 46.8% were also EBNA-1 DNA positive. Viral loads (combined targets) were detectable between 2.0 × 102 to 6.2 × 104 copies/ml of whole blood, but 54.2% and 85.1% of samples were below 2.0 × 103 copies/ml for EBNA-1 and BHRF-1 DNA levels, respectively. All samples with measurable EBV DNA were EBV VCA IgG antibody positive, which were found in 95.9% of the population sample. There was a modest correlation between VCA IgG antibody titres and BHRF-1 DNA load (Spearman's rho = 0.13, p = 0.05) and a weaker (not statistically significant) correlation between EBNA-1 DNA load and VCA IgG antibody titres (Spearman's rho = 0.11, p = 0.11) (Table 4).

Table 4 EBV DNA load and antibody titre detection rates in the population samples (Group 5, n = 218).

Discussion

With increasing availability of nucleic acid testing (NAT) methods, measuring EBV DNA in blood has proven valuable in diagnosing and monitoring PTLD [16, 21, 22, 37–41], NPC [42, 43], IM [13, 44], EBV infection in HIV-infected individuals [8, 13, 45], BL [13] and chronic active EBV infection [18, 46]. In this study, we successfully developed two in-house QPCR methods incorporating a novel single quantification standard containing two EBV DNA targets for measuring viral load on the Rotor-Gene 6000™. Substituting SYBR Green I dye as a fluorescent marker for product accumulation over fluorogenic probes, this method proved useful for quantifying EBV DNA concentrations in clinical samples from individuals with a variety of EBV-associated disorders or immune dysfunctions and in a healthy population sample.

Previous studies in PTLD have found that EBV DNA loads increased with disease progression and decreased with remission of lymphoproliferation [47, 48]. This pattern was observed in Group 1, where EBV DNA loads appeared to be correlated with disease status. We found similar EBV DNA loads to those previously reported, with most studies showing EBV DNA concentrations ranging from 5.0 × 102 to 2.0 × 107 copies/ml in whole blood, plasma and serum [37, 49, 50]. EBV DNA was also detected in CSF at concentrations comparable to plasma, however detectable CSF EBV DNA has been previously reported only in association with acquired immunodeficiency syndrome (AIDS)-related brain lymphoma [51]. The significance of EBV DNA in CSF of PTLD remains to be elucidated.

EBV DNA loads in IM patients were also similar to those reported in the literature [13, 22, 26, 44, 52], although some authors described loads as high as 106 and 107 copies/ml [12, 46, 53]. In Group 3, EBV DNA loads were consistent with acute phase EBVAHS [46, 54], and correlated with the deterioration of the patient's disease condition. Elazary et al also found that a viral load ranging from 104-105 copies/ml was associated with poor patient outcome [54]. One study found much higher EBV DNA loads (up to 107 copies/ml) [55], but this may have been due to differences in sample type and detection methods. In Group 4, EBV DNA was detected in 33% of samples (22% of plasma, 67% of PBMC), compared to 34% to 76% positivity reported in other studies [8, 26]. Notably however, these studies used whole blood for quantifying EBV DNA load, which could have increased the probability of viral DNA detection. As none of the Group 4 patients were known to have EBV-related disease, low positivity ratios and viral loads were expected.

Similar to our findings, the literature describes EBV DNA detectable from 102 to 104 copies/ml and positivity ratios up to 29% in whole blood of healthy individuals [11–13, 26, 38, 56–59]. However, DNA loads as high as 5.5 × 105 copies/ml of whole blood and a positivity ratio of 72% have been reported [58]. Differences in the results may be attributable to more sensitive methods associated with nested PCR and dual-labelled probes [58]. Interestingly, another study showed 100% EBV DNA positivity in whole blood, although DNA loads were all below the detection limit of the assay (2.0 × 103 copies/ml) [38].

In the population sample the EBV VCA IgG antibody detection rate was consistent with levels of EBV sero-positivity in Western societies [2]. One study previously showed a correlation between EBV VCA IgG antibody titres and EBV viral load (detectable versus non-detectable) [60]. We similarly found a modest correlation with quantitative BHRF-1 DNA loads, and a weaker (not statistically significant) correlation with EBNA-1 DNA load (see Table 4).

We noted some discrepancies in our measures of EBV positivity. In one PTLD patient (Patient D), plasma was qualitative EBV PCR negative whilst simultaneously reporting an EBV DNA load of 1.3 × 108 copies/ml in whole blood. However, a growing number of studies have shown that cell-associated EBV is detectable before plasma EBV DNA and can persist without accompanying plasma DNA loads [21, 48]. In Group 2, Patient G, despite being EBV VCA IgM antibody positive, was EBV QPCR negative. As EBV DNA loads can change rapidly from being undetectable to being very high in a short period of time [38], it is possible that sampling occurred late in the convalescent phase where low EBV DNA positivity ratios of 44% have been previously reported [46]. Other factors contributing to DNA load variation include differences in sample type, method of extraction or NAT, and target chosen for PCR amplification.

As specimen type is known to influence DNA loads and impact on assay performance [36], unfractionated EDTA whole blood was used for DNA quantification where possible. The dynamic changes of EBV DNA are better reflected in circulating whole blood [38], which also contains all the compartments that may harbour virus [13, 21, 61]. However, despite reports of greater test sensitivity with whole blood [12, 36], EBV DNA load has also been quantified in PBMC [14, 16, 62–64]. Although infection is typically associated with cell compartments [8, 12, 13], EBV DNA is also found in cell-free blood partitions such as plasma or serum, usually in fragmented, cell-derived form [12]. In this study, 2 of 9 plasma samples from HIV-infected patients had detectable EBV DNA, compared to 2 out of 3 PBMC samples. As we did not have simultaneous plasma and PBMC samples from the same individuals, we were unable to assess the differences in viral load between these compartments. Further studies comparing suitability of different sample types in various EBV-related diseases and immune disorders are required.

The method of DNA purification is known to affect viral load measurements. One study showed yield from manually extracted DNA was 57% higher than that of robotic systems [65]. Therefore, to improve DNA recovery and maximise PCR sensitivity, samples here were purified using a commercial silica-based column method [61, 66]. For optimal quantitation results, an earlier study showed that DNA should be subjected to PCR within one to two weeks post-extraction [67]. Here, delay between extraction and testing could have contributed to low DNA loads and positivity ratios in clinical samples. Furthermore, DNA from blood samples that had undergone more than four freeze-thaw cycles were found to be partially degraded [68]. Since the clinical samples used here were tested retrospectively, monitoring these conditions were not possible.

EBV DNA loads also vary according to type and size of gene target [69]. Ryan et al, found assay sensitivity was dependent on the specific gene segment and that different targets had varying lower limits of detection [15]. For EBV, BamHI-W is reportedly 10 times more sensitive than other targets for PCR, allowing for detection of viral DNA at trace amounts [8, 13, 15]. However, precise quantification of viral genomes is complicated by the number of reiterated BamHI-W sequences among EBV strains, which typically ranges between 7 and 11 repeats per genome [15]. To avoid overestimation in this study, we chose to use the next most sensitive EBV gene; EBNA-1 [15], and an abundantly expressed gene, BHRF-1, for QPCR.

Despite targeting highly conserved EBV regions, selective drop out of amplifiable EBV DNA at the EBNA-1 and BHRF-1 loci was observed in Group 4 (Patients N and X), and in 25 of 218 (11.5%) whole bloods from the population sample. Instead of amplifying both EBV DNA genes, only one target was detected, 93% of which had viral loads less than 2.0 × 103 copies/ml. As beta-globin was detected in all samples, PCR inhibitors and/or defective nucleic acid purification methods were excluded [70]. Alternatively, selective drop out may have been due to low viral load and/or sampling error [71]. Since load determination is reliant on the amount of EBV genomes pipetted into a reaction and assumes viral homogeneity, QPCR results, particularly at low viral load levels are prone to random sampling error. This phenomenon is well documented in DNA quantification and results in less reliable viral load measurements [70, 71]. Therefore, samples reporting low levels of target nucleic acid may not be reproducible in repeated assays from the same or different specimens [72].

Currently, there are no standardised methods for measuring EBV DNA, complicating inter-laboratory comparisons in multicentre studies of EBV-related diseases. Standardisation is difficult as PCR assay conditions vary between laboratories, leading to variations in the accuracy and reproducibility of viral load quantification [21]. Although there appears to be a strong concordance between laboratories for qualitative EBV DNA estimates, there continues to be marked inconsistency in quantitative results [73]. It has been suggested that the use of unfractionated whole blood [26] or an international calibration standard could be the first step towards standardisation [73]. However, instrumentation, chemistries, gene targets and other test-related aspects remain diverse. One solution for enabling inter-laboratory comparisons is the distribution of proficiency panels such as QCMD. Such programs have already been used for assessing methods for the detection and quantification of EBV and other viruses [27, 74, 75].

Conclusion

This is the first reported study that uses the SYBR Green I dye on the Rotor-gene 6000â„¢ with a novel quantification standard containing two EBV targets for measuring EBV DNA load. The assays proved successful in the quantification of EBV genomes in clinical cases and should be considered as a cost effective and sensitive PCR alternative to probe-based assays. This approach can be modified to detect and quantify other latent herpesviruses such as HHV6, CMV, and VZV. This procedure is suitable for robotics and automation, and would be a useful addition in larger laboratories.

References

  1. Steven NM: Epstein-Barr virus latent infection in vivo. Rev Med Virol 1997,7(2):97-106. 10.1002/(SICI)1099-1654(199707)7:2<97::AID-RMV190>3.0.CO;2-M

    Article  PubMed  CAS  Google Scholar 

  2. Hess RD: Routine Epstein-Barr virus diagnostics from the laboratory perspective: still challenging after 35 years. J Clin Microbiol 2004,42(8):3381-7. 10.1128/JCM.42.8.3381-3387.2004

    Article  PubMed  PubMed Central  Google Scholar 

  3. Tsai DE, et al.: EBV PCR in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: results of a two-arm prospective trial. Am J Transplant 2008,8(5):1016-24. 10.1111/j.1600-6143.2008.02183.x

    Article  PubMed  CAS  Google Scholar 

  4. Loginov R, et al.: Monitoring of EBV-DNAemia by quantitative real-time PCR after adult liver transplantation. J Clin Virol 2006,37(2):104-108. 10.1016/j.jcv.2006.06.012

    Article  PubMed  CAS  Google Scholar 

  5. Timbury MC, Edmond E: Herpesviruses. J Clin Pathol 1979,32(9):859-81. 10.1136/jcp.32.9.859

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. Cohen JI: Epstein-Barr virus infection. N Engl J Med 2000,343(7):481-92. 10.1056/NEJM200008173430707

    Article  PubMed  CAS  Google Scholar 

  7. Kimura H, et al.: Viral load in Epstein-Barr virus-associated hemophagocytic syndrome. Microbiol Immunol 2002,46(8):579-82.

    Article  PubMed  CAS  Google Scholar 

  8. Stevens SJ, et al.: High Epstein-Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy and quantitative EBV serology. AIDS 2002,16(7):993-1001. 10.1097/00002030-200205030-00005

    Article  PubMed  CAS  Google Scholar 

  9. Espy MJ, et al.: Comparison of three methods for extraction of viral nucleic acids from blood cultures. J Clin Microbiol 1995,33(1):41-4.

    PubMed  CAS  PubMed Central  Google Scholar 

  10. Leung E, et al.: Dynamic EBV gene loads in renal, hepatic, and cardiothoracic transplant recipients as determined by real-time PCR light cycler. Transpl Infect Dis 2004,6(4):156-64. 10.1111/j.1399-3062.2004.00073.x

    Article  PubMed  CAS  Google Scholar 

  11. Bai X, et al.: Quantitative polymerase chain reaction for human herpesvirus diagnosis and measurement of Epstein-Barr virus burden in posttransplant lymphoproliferative disorder. Clin Chem 1997,43(10):1843-9.

    PubMed  CAS  Google Scholar 

  12. Kozic S, et al.: Evaluation of a commercial real-time PCR assay for quantitation of Epstein-Barr virus DNA in different groups of patients. J Virol Methods 2006,135(2):263-8. Epub 2006 May 2 10.1016/j.jviromet.2006.03.013

    Article  PubMed  CAS  Google Scholar 

  13. Stevens SJ, Pronk I, Middeldorp JM: Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen. J Clin Microbiol 2001,39(4):1211-6. 10.1128/JCM.39.4.1211-1216.2001

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  14. Leung E, et al.: Use of real-time PCR to measure Epstein-Barr virus genomes in whole blood. J Immunol Methods 2002,270(2):259-67.

    Article  PubMed  CAS  Google Scholar 

  15. Ryan JL, et al.: Epstein-Barr virus quantitation by real-time PCR targeting multiple gene segments: a novel approach to screen for the virus in paraffin-embedded tissue and plasma. J Mol Diagn 2004,6(4):378-85.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  16. Rowe DT, et al.: Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders. J Clin Microbiol 1997,35(6):1612-5.

    PubMed  CAS  PubMed Central  Google Scholar 

  17. Fan H, Gulley ML: Epstein-Barr viral load measurement as a marker of EBV-related disease. Mol Diagn 2001,6(4):279-89.

    Article  PubMed  CAS  Google Scholar 

  18. Kimura H, et al.: Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay. J Clin Microbiol 1999,37(1):132-6.

    PubMed  CAS  PubMed Central  Google Scholar 

  19. Lo YM, et al.: Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999,59(21):5452-5.

    PubMed  CAS  Google Scholar 

  20. Lo YM, et al.: Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res 1999,59(6):1188-91.

    PubMed  CAS  Google Scholar 

  21. Stevens SJ, et al.: Role of Epstein-Barr virus DNA load monitoring in prevention and early detection of post-transplant lymphoproliferative disease. Leuk Lymphoma 2002,43(4):831-40. 10.1080/10428190290016971

    Article  PubMed  Google Scholar 

  22. Niesters HGM, et al.: Development of a real-time quantitative assay for detection of Epstein-Barr virus. J Clin Microbiol 2000,38(2):712-5.

    PubMed  CAS  PubMed Central  Google Scholar 

  23. Xu S, et al.: A comparison of quantitative-competitive and realtime PCR assays using an identical target sequence to detect Epstein-Barr virus viral load in the peripheral blood. J Virol Methods 2006,137(2):205-12. 10.1016/j.jviromet.2006.06.012

    Article  PubMed  CAS  Google Scholar 

  24. Brengel-Pesce K, et al.: Routine use of real-time quantitative PCR for laboratory diagnosis of Epstein-Barr virus infections. J Med Virol 2002,66(3):360-9. 10.1002/jmv.2153

    Article  PubMed  CAS  Google Scholar 

  25. Mackay IM, Arden KE, Nitsche A: Real-time PCR in virology. Nucleic Acids Res 2002,30(6):1292-305. 10.1093/nar/30.6.1292

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  26. Fafi-Kremer S, et al.: Evaluation of the Epstein-Barr virus R-gene quantification kit in whole blood with different extraction methods and PCR platforms. J Mol Diagn 2008,10(1):78-84. 10.2353/jmoldx.2008.070054

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Ruiz G, et al.: Comparison of commercial real-time PCR assays for quantification of Epstein-Barr virus DNA. J Clin Microbiol 2005,43(5):2053-7. 10.1128/JCM.43.5.2053-2057.2005

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  28. Papin JF, Vahrson W, Dittmer DP: SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability. J Clin Microbiol 2004,42(4):1511-8. 10.1128/JCM.42.4.1511-1518.2004

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Karlsen F, Steen HB, Nesland JM: SYBR green I DNA staining increases the detection sensitivity of viruses by polymerase chain reaction. J Virol Methods 1995,55(1):153-6. 10.1016/0166-0934(95)00053-W

    Article  PubMed  CAS  Google Scholar 

  30. Hilscher C, Vahrson W, Dittmer DP: Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability. Nucleic Acids Res 2005,33(21):e182.. 10.1093/nar/gni181

    Article  PubMed  PubMed Central  Google Scholar 

  31. Lucas RM, et al.: Associations between silicone skin cast score, cumulative sun exposure, and other factors in the ausimmune study: a multicenter Australian study. Cancer Epidemiol Biomarkers Prev 2009,18(11):2887-94. 10.1158/1055-9965.EPI-09-0191

    Article  PubMed  CAS  Google Scholar 

  32. Humme S, et al.: The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold. Proc Natl Acad Sci USA 2003,100(19):10989-94. 10.1073/pnas.1832776100

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  33. Lee MA, Diamond ME, Yates JL: Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by Epstein-Barr virus. J Virol 1999,73(4):2974-82.

    PubMed  CAS  PubMed Central  Google Scholar 

  34. Henderson S, et al.: Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc Natl Acad Sci USA 1993,90(18):8479-83. 10.1073/pnas.90.18.8479

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  35. Huang Y, et al.: Proper developmental control of human globin genes reproduced by transgenic mice containing a 160-kb BAC carrying the human beta-globin locus. Blood Cells Mol Dis 2000,26(6):598-610. 10.1006/bcmd.2000.0339

    Article  PubMed  CAS  Google Scholar 

  36. Hakim H, et al.: Comparison of various blood compartments and reporting units for the detection and quantification of Epstein-Barr virus in peripheral blood. J Clin Microbiol 2007,45(7):2151-5. 10.1128/JCM.02308-06

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. van Esser JW, et al.: Molecular quantification of viral load in plasma allows for fast and accurate prediction of response to therapy of Epstein-Barr virus-associated lymphoproliferative disease after allogeneic stem cell transplantation. Br J Haematol 2001,113(3):814-21. 10.1046/j.1365-2141.2001.02789.x

    Article  PubMed  CAS  Google Scholar 

  38. Stevens SJ, et al.: Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients. Blood 2001,97(5):1165-71. 10.1182/blood.V97.5.1165

    Article  PubMed  CAS  Google Scholar 

  39. Lucas KG, et al.: Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation. Blood 1998,91(10):3654-61.

    PubMed  CAS  Google Scholar 

  40. Kimura H, et al.: Measuring Epstein-Barr virus (EBV) load: the significance and application for each EBV-associated disease. Rev Med Virol 2008,18(5):305-19. 10.1002/rmv.582

    Article  PubMed  CAS  Google Scholar 

  41. Wheless SA, et al.: Post-transplantation lymphoproliferative disease: Epstein-Barr virus DNA levels, HLA-A3, and survival. Am J Respir Crit Care Med 2008,178(10):1060-5. 10.1164/rccm.200804-531OC

    Article  PubMed  CAS  Google Scholar 

  42. Chan KC, et al.: Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients. Cancer Res 2003,63(9):2028-32.

    PubMed  CAS  Google Scholar 

  43. Lin JC, et al.: Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 2004,350(24):2461-70. 10.1056/NEJMoa032260

    Article  PubMed  CAS  Google Scholar 

  44. Bauer CC, et al.: Serum Epstein-Barr virus DNA load in primary Epstein-Barr virus infection. J Med Virol 2005,75(1):54-8. 10.1002/jmv.20237

    Article  PubMed  Google Scholar 

  45. Dehee A, et al.: Quantification of Epstein-Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR. J Med Virol 2001,65(3):543-52. 10.1002/jmv.2071

    Article  PubMed  CAS  Google Scholar 

  46. Yamamoto M, et al.: Detection and quantification of virus DNA in plasma of patients with Epstein-Barr virus-associated diseases. J Clin Microbiol 1995,33(7):1765-8.

    PubMed  CAS  PubMed Central  Google Scholar 

  47. Wagner HJ, et al.: Patients at risk for development of posttransplant lymphoproliferative disorder: plasma versus peripheral blood mononuclear cells as material for quantification of Epstein-Barr viral load by using real-time quantitative polymerase chain reaction. Transplantation 2001,72(6):1012-9. 10.1097/00007890-200109270-00006

    Article  PubMed  CAS  Google Scholar 

  48. Rowe DT, et al.: Epstein-Barr virus load monitoring: its role in the prevention and management of post-transplant lymphoproliferative disease. Transpl Infect Dis 2001,3(2):79-87. 10.1034/j.1399-3062.2001.003002079.x

    Article  PubMed  CAS  Google Scholar 

  49. Rosselet A, et al.: Associations of serum EBV DNA and gammopathy with post-transplant lymphoproliferative disease. Clin Transplant 2009,23(1):74-82. 10.1111/j.1399-0012.2008.00904.x

    Article  PubMed  Google Scholar 

  50. Wada K, et al.: Simultaneous quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus 6 DNA in samples from transplant recipients by multiplex real-time PCR assay. J Clin Microbiol 2007,45(5):1426-32. 10.1128/JCM.01515-06

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  51. Bossolasco S, et al.: Epstein-Barr virus DNA load in cerebrospinal fluid and plasma of patients with AIDS-related lymphoma. J Neurovirol 2002,8(5):432-8. 10.1080/13550280260422730

    Article  PubMed  CAS  Google Scholar 

  52. Stevens SJ, et al.: Monitoring of epstein-barr virus DNA load in peripheral blood by quantitative competitive PCR. J Clin Microbiol 1999,37(9):2852-7.

    PubMed  CAS  PubMed Central  Google Scholar 

  53. van Laar JA, et al.: Epstein-Barr viral load assessment in immunocompetent patients with fulminant infectious mononucleosis. Arch Intern Med 2002,162(7):837-9. 10.1001/archinte.162.7.837

    Article  PubMed  Google Scholar 

  54. Elazary AS, et al.: Severe Epstein-Barr virus-associated hemophagocytic syndrome in six adult patients. J Clin Virol 2007,40(2):156-9. 10.1016/j.jcv.2007.06.014

    Article  PubMed  Google Scholar 

  55. Teramura T, et al.: Quantitative analysis of cell-free Epstein-Barr virus genome copy number in patients with EBV-associated hemophagocytic lymphohistiocytosis. Leuk Lymphoma 2002,43(1):173-9. 10.1080/10428190210176

    Article  PubMed  CAS  Google Scholar 

  56. Compston LI, et al.: Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions. J Virol Methods 2008,151(1):47-54. 10.1016/j.jviromet.2008.03.023

    Article  PubMed  CAS  Google Scholar 

  57. Kullberg-Lindh C, et al.: Comparison of serum and whole blood levels of cytomegalovirus and Epstein-Barr virus DNA. Transpl Infect Dis 2008,10(5):308-15. 10.1111/j.1399-3062.2008.00313.x

    Article  PubMed  CAS  Google Scholar 

  58. Hudnall SD, et al.: Herpesvirus prevalence and viral load in healthy blood donors by quantitative real-time polymerase chain reaction. Transfusion 2008,48(6):1180-7. 10.1111/j.1537-2995.2008.01685.x

    Article  PubMed  Google Scholar 

  59. Engelmann I, et al.: Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 DNA in blood and other clinical specimens. J Med Virol 2008,80(3):467-77. 10.1002/jmv.21095

    Article  PubMed  CAS  Google Scholar 

  60. Besson C, et al.: Positive correlation between Epstein-Barr virus viral load and anti-viral capsid immunoglobulin G titers determined for Hodgkin's lymphoma patients and their relatives. J Clin Microbiol 2006,44(1):47-50. 10.1128/JCM.44.1.47-50.2006

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  61. Fafi-Kremer S, et al.: Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma. J Clin Virol 2004,30(2):157-64. 10.1016/j.jcv.2003.10.002

    Article  PubMed  CAS  Google Scholar 

  62. Jabs WJ, et al.: Normalized quantification by real-time PCR of Epstein-Barr virus load in patients at risk for posttransplant lymphoproliferative disorders. J Clin Microbiol 2001,39(2):564-9. 10.1128/JCM.39.2.564-569.2001

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  63. Wadowsky RM, et al.: Measurement of Epstein-Barr virus DNA loads in whole blood and plasma by TaqMan PCR and in peripheral blood lymphocytes by competitive PCR. J Clin Microbiol 2003,41(11):5245-9. 10.1128/JCM.41.11.5245-5249.2003

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  64. Baldanti F, et al.: Kinetics of Epstein-Barr virus DNA load in different blood compartments of pediatric recipients of T-cell-depleted HLA-haploidentical stem cell transplantation. J Clin Microbiol 2008,46(11):3672-7. 10.1128/JCM.00913-08

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  65. Riemann K, et al.: Comparison of manual and automated nucleic acid extraction from whole-blood samples. J Clin Lab Anal 2007,21(4):244-8. 10.1002/jcla.20174

    Article  PubMed  CAS  Google Scholar 

  66. Schuurman T, et al.: Reduced PCR sensitivity due to impaired DNA recovery with the MagNA Pure LC total nucleic acid isolation kit. J Clin Microbiol 2005,43(9):4616-22. 10.1128/JCM.43.9.4616-4622.2005

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  67. Malcomson RD, et al.: The scope of quantitative polymerase chain reaction assays in clinical molecular pathology. Clin Mol Pathol 1995,48(4):M178-M183. 10.1136/mp.48.4.M178

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  68. Lahiri DK, Schnabel B: DNA isolation by a rapid method from human blood samples: effects of MgCl2, EDTA, storage time, and temperature on DNA yield and quality. Biochem Genet 1993,31(7-8):321-8. 10.1007/BF02401826

    Article  PubMed  CAS  Google Scholar 

  69. Stevens SJ, et al.: Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia. J Clin Microbiol 2005,43(7):3066-73. 10.1128/JCM.43.7.3066-3073.2005

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  70. Gulley ML, Fan H, Elmore SH: Validation of Roche LightCycler Epstein-Barr virus quantification reagents in a clinical laboratory setting. J Mol Diagn 2006,8(5):589-97. 10.2353/jmoldx.2006.050152

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  71. Diaco D: Practical Considerations for the Design of Quantitative PCR Assays. In PCR Strategies. Edited by: G DH, S JJ, Innis MA. Academic Press Limited, London; 1995:84-107. full_text

    Chapter  Google Scholar 

  72. Smith TF, et al.: Quantitative real-time polymerase chain reaction for evaluating DNAemia due to cytomegalovirus, Epstein-Barr virus, and BK virus in solid-organ transplant recipients. Clin Infect Dis 2007,45(8):1056-61. 10.1086/521909

    Article  PubMed  CAS  Google Scholar 

  73. Hayden RT, et al.: Multicenter comparison of different real-time PCR assays for quantitative detection of Epstein-Barr virus. J Clin Microbiol 2008,46(1):157-63. 10.1128/JCM.01252-07

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  74. Perandin F, et al.: Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma. BMC Microbiol 2007, 7: 22. 10.1186/1471-2180-7-22

    Article  PubMed  PubMed Central  Google Scholar 

  75. Pillet S, Bourlet T, Pozzetto B: Comparative evaluation of a commercially available automated system for extraction of viral DNA from whole blood: application to monitoring of epstein-barr virus and cytomegalovirus load. J Clin Microbiol 2009,47(11):3753-5. 10.1128/JCM.01497-09

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The Ausimmune Investigator Group includes C Chapman, A Coulthard, K Dear, T Dwyer, T Kilpatrick, R Lucas, T McMichael, MP Pender, A-L Ponsonby, B Taylor, P Valery, I van der Mei and D Williams. The Ausimmune Study is funded by the National Multiple Sclerosis Society of the USA, the National Health & Medical Research Council (Project Grant 316901) and Multiple Sclerosis Research Australia. We also acknowledge the work of the Ausimmune Study research nurses who undertook sample collection: S Agland, B Alexander, M Davis, Z Dunlop, A Wright, R Scott, J Selvidge, M Steele, K Turner, B Wood and the study project officers, H Rodgers and C Jozwick. Clinical samples were kindly provided by N Gilroy, D Gottlieb, P Ferguson, F Kwok and I Kay. We would also like to thank B Wang of the Westmead Millennium Institute for assisting with the cloning work, B O'Toole for statistical analyses, D Patel for assistance with the serology and C Toi for laboratory guidance and review of the manuscript.

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Authors' contributions

MLL developed the assays, carried out all of the DNA work, assisted in the data analysis and result interpretation, and writing of the manuscript. On behalf of the Ausimmune Investigator group, RML supplied the whole blood and serum from the population sample, and was involved in the data analysis. VMR aided in primer design and JT performed the serology testing. MLL, DED, VMR, RML and ALP were involved in the design and conception of the study. All authors have read, reviewed and approved the final manuscript.

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Lay, ML.J., Lucas, R.M., Ratnamohan, M. et al. Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye. Virol J 7, 252 (2010). https://doi.org/10.1186/1743-422X-7-252

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